Transforming Growth Factor xcex2 (TGF-xcex2) is a potent growth regulatory protein and a key molecule implicated in various fibrotic (scarring) disorders. Most of the cells secrete TGF-xcex21 in a predominantly inactive high molecular weight form, latent TGF-xcex2 (L-TGF-xcex2). Latent TGF-xcex2 is composed of an amino-terminal latency-associated peptide (LAP) noncovalently associated with the carboxyl-terminal mature TGF-xcex2. The latency-associated peptide, is disulfide-bonded to a second, structurally unrelated protein, latent TGF-xcex2 binding protein (LTBP), which plays a role in the processing and secretion of TGF-xcex21 (1).
A major mechanism of regulating TGF-xcex2 activity occurs through factors which control the processing of the latent to biologically active form of the molecule. Physiochemical activation can occur by extremes of pH, heat, chaotropic agents (sodium dodecyl sulfate, urea) and deglycosylation (2, 3, 4, 5). Activation in vivo is more complex and not well understood.
Cell-mediated activation was first achieved by co-cultures of either pericytes or smooth muscle cells with capillary endothelial cells (6, 7). This method requires interaction of two cell types from the same species. The activation is apparently mediated by plasmin since activation is blocked by plasmin/serine protease inhibitors (8). Cellular activation is thought to require binding of the latent TGF-xcex2 to the mannose-6-phosphate/insulin-like growth factor II receptor (9) via the mannose-6-phosphate molecules found on the LAP component of the latent form of TGF-xcex2 (10). Tissue type II transglutaminase has also been demonstrated as a requirement for activation in this cell-dependent model which may function to crosslink latent TGF-xcex2 to the matrix molecules (11). The final requirement for activation involves the LTBP. It has been proposed that LTBP is necessary for concentrating the latent TGF-xcex2 complex onto the cell surface where it is subsequently activated, presumably by tissue transglutaminase and/or plasmin (12). Three different LTBP""s have been identified and cloned (13-15) which may indicate a potential mechanism by which cells may control interactions of the latent TGF-xcex2 complexes with specific tissue sites or cell types. In addition, there are reports of TGF-xcex2 activation occurring independently of these mechanisms by binding of the latent complex to thrombospondin, an extracellular matrix associated glycoprotein (16, 17). Although L-TGF-xcex2 is not activated under normal culture conditions unless co-cultures are prepared, cells in homotypic cultures can be induced to form active TGF-xcex2 by application of specific agents. Among these agents are retinoids, which induce the activation of latent-TGF-xcex21 in keratinocytes, endothelial cells and osteoclasts (18, 19, 20). Retinoid-induced activation of TGF-xcex21 is dependent upon plasmin (18, 19). Anti-estrogens (tamoxifen or toremifine) induced the production of active TGF-xcex2 in fetal fibroblasts and mammary carcinoma cells (21). Exposure of bovine arterial or capillary endothelial cells to bFGF (basic Fibroblast Growth Factor) in vitro also resulted in activation of TGF-xcex2 apparently by a plasmin-dependent mechanism (22). Growth zone costochondral chondrocyte matrix vesicles were able to activate latent TGF-xcex2 when incubated with 1,25-dihydroxy vitamin D3 through a direct action of the vitamin on the extracellular matrix vesicle membrane (23). In addition there are reports that small quantities of active TGF-xcex21 could be detected in conditioned media from human prostate epithelial cells (24), melanoma cell lines (25) and glioblastoma cells (26); however the mechanism of this activation is not understood.
To date, none of the systems generating active TGF-xcex21 has been utilized as a screening model for agents with the potential ability to sustain the latency of TGF-xcex21. The co-culture method has been studied extensively and the mechanisms responsible for activation of TGF-xcex21 in this system were elucidated. However, this co-culture method lacks reproducibility and has a strong requirement for screening large numbers of cell clones to produce an effective system.
The aim of these studies was to develop a new in vitro model for TGF-xcex2 activation for subsequent screening of molecules with potential abilities to interfere with its activation. Since TGF-xcex2 has been shown to be a key factor in scarring and fibrotic disorders, agents shown to be active in such an assay would be expected to function as anti-fibrotics and anti-scarring in vivo.
A novel method has been developed for screening and identifying modulators of scar formation, such as anti-scarring and anti-fibrotic agents. This method offers simplicity, it is reproducible and could be adopted to screen large number of modulator compounds to identify new potential anti-fibrotic agents.
This method has characteristics in common with the bovine arterial endothelial cells/bovine arterial smooth muscle cells (BAEC/BASMC) co-culture system, but is more sensitive and does not require screening large number of clonal lines for developing an effective method.
In this novel system, similar to the co-culture system, activation of L-TGF-xcex21 occurs by several independent mechanisms which involve binding of the latent complex to mannose-6 phosphate/insulin-like growth factor-II (M6P/IGF-II) receptors, thrombospondin and/or tissue type II transglutaminase. But, in contrast to the co-culture system, this macrophage-dependent system does not appear to involve plasmin.
Using this method, potential novel anti-fibrotic agents were identified, such as insulin-like growth factor II defined as IGF-II, (used separately or in combination with insulin-like growth factor binding protein-2 (IGFBP-2) as a delivery vehicle), tissue type II transglutaminase inhibitors and anti-inflammatory agents (such as hydrocortisone).
A novel mechanism of action for M6P has been proposed herein which is based on downregulation of M6P/IGF-II receptor and TGF-xcex21 mRNAs.